Influence of various temperatures, seed priming treatments and durations on germination and growth of the medicinal plant Aspilia africana

For millennia, Aspilia africana has been used across Africa to treat various diseases including malaria, wounds, and diabetes. In this study, temperature influenced the in vitro germination of A. africana with highest final germination percentage (FGP) and germination index (GI) of 65.0 ± 7.64% and 2.26 ± 0.223, respectively, at 19.8 °C. Priming seeds with H2O, KNO3, and GA3 (gibberellic acid 3) improved both in vitro germination and ex vitro emergence of A. africana seeds. Seed priming with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1.44 \times 10^{ - 3 }$$\end{document}1.44×10-3 M GA3 produced overall highest in vitro FGP (from 90.0 ± 4.08% to 100 ± 0.00%) and GI (from 2.97 ± 0.385 to 3.80 ± 0.239) across all priming durations. Seeds primed with KNO3 had better germination parameters for 6 and 12 h compared to 18 and 24 h. Furthermore, the highest in vitro FGP (100 ± 0.00%) was observed in seeds primed for 12 h with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1.44 \times 10^{ - 3 }$$\end{document}1.44×10-3 M GA3. Ex vitro A. africana seed emergence was significantly enhanced by GA3 priming. Priming A. africana seeds with H2O, KNO3, and GA3 improved their growth after 3 months, with the overall best growth for seeds primed with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2.89 \times 10^{ - 4 }$$\end{document}2.89×10-4 M GA3. Seed priming of A. africana is a feasible approach for improving germination and seed emergence, and enhancing plant growth.


Experiment 1: effect of temperature on in vitro germination of A. africana seeds. A. africana
seeds were placed using sterile forceps in crystal-grade polystyrene Petri dishes (100 × 20 mm) containing filter papers moistened with 5 ml distilled water. Each Petri dish contained 10 seeds with six replicates for each temperature, ranging from 17.6 to 27.5 °C with an increment of 1.1 °C, and dishes were placed on a thermogradient germinator chamber under dark conditions (Fig. 1b).
The seeds were monitored for germination every 24 h for 15 d. The Petri dishes with seeds were only exposed to light for a short time during the counting of the germinated seeds. Seeds with a minimum of 2 mm radicle length were counted as germinated. The germination parameters considered for determining the effects of temperature on seed germination of A. africana were final germination percentage (FGP), germination index (GI), mean germination rate (MGR), and time required for 50% germination (T 50 ). The same parameters were used to investigate the effects of priming treatments and durations on the in vitro and ex vitro germination of A. africana seeds. The following formulae were used to calculate the germination parameters: FGP = N Nt × 100 (N is the number of germinated seeds at the final count; Nt is the total number of seeds in the Petri dish); GI = ∑ (Gt/ Dt) (Gt is the number of germinated seeds on day t, and Dt is time corresponding to Gt in days); and MGR = ( n) (nt) (n is the number of newly germinated seeds at time t, and t is the number of days from planting). T 50 was calculated according to the formula modified from Farooq, et al. 19 , T 50 = t i + N 2 −n i (tj−ti) n j −n i (N is the final number of germinated seeds, n i and n j are the cumulative number of germinated seeds counted at time t i and t j , respectively, when n i < N 2 < n j ).

Experiment 2: effect of priming duration and priming treatments on in vitro germination of
A. africana seeds. Sterilized A. africana seeds were primed at varying times of 6 h, 12 h, 18 h, and 24 h in different priming solutions, such as distilled water (hydro-priming) and potassium nitrate (halo-priming) at different concentrations (0.1, 0.5, and 1 M) and gibberellic acid-3 (GA 3 ; hormo-priming) at concentrations of 2.89 × 10 −5 , 2.89 × 10 −4 , and 1.44 × 10 −3 M. For priming treatments, 60 seeds were placed in 5 ml of each priming solution in crystal-grade polystyrene Petri dishes (100 × 40 mm). Upon completion of the treatment, the primed seeds were rinsed three times with sterile water, blotted, and dried back to their near initial weight at ambient temperature. The seeds were then placed in crystal-grade polystyrene Petri dishes (100 × 20 mm) containing filter paper saturated with autoclaved distilled water. Each Petri dish (100 × 20 mm) contained 10 seeds with five replicates for each treatment. The Petri dishes containing the seeds were kept in a thermogradient germinator chamber maintained at 19.8 °C (determined to be ideal from experiment 1) in darkness. Nonprimed A. africana seeds were used as a control. Seeds showing signs of fungal contamination were removed from the Petri dishes. The seeds were monitored for germination every 24 h for 15 d and were only exposed to light for a short time during the counting of germinated seeds. Seeds with a minimum of 2 mm radicle length were counted as germinated. The same parameters and formulae used to assess germination in Experiment 1 were used in Experiments 2 and 3. A. africana seeds, the priming procedure used in Experiment 2 was repeated, except that seeds were treated with the priming solutions for a uniform priming duration of 12 h. The primed seeds were then planted in an autoclaved mixture of horticulture soil (consisting of about 40% mineral soil, 10% organic matter and 50% pore space filled by water and air) containing perlite (Kyungdong ONE Co. Ltd, Republic of Korea), one of the natural volcanic aluminosilicate glasses 20 and peat pellet soil (Jiffy-7, 33 mm from Jiffy Products International AS, Norway) in a 1:1 ratio (determined as an ideal composition for the growth of A. africana 18 ) in a plastic planting tray (30 × 25 × 10 cm). Twenty seeds of A. africana were planted in each tray at a depth of 1 cm and a distance of 5 cm from each other. Each treatment was replicated three times. The seeds in the soil were watered and the trays were kept in growth chambers maintained at 19.8 ± 1 °C for a 16 h photoperiod. Light intensity was maintained at 33.73 µmol/m2/s using cool white fluorescent tubes. The relative humidity in the growth chamber was maintained at 70%. The seeds in the tray were watered every two days until the end of the experiment. The number of seeds germinated every 24 h was counted and recorded for each treatment until no further emergence occurred. Seeds were counted as emerged when the hypocotyl length was at least 3 mm. The FGP, MGR, T 50 , and GI were calculated. To determine the effects of different priming treatments on the early growth of A. africana, seedlings from the differently primed seeds were uniformly re-spaced and allowed to continue growing in the growth chambers, and the growth rates were determined after three months. Each planting tray was carefully immersed in water to soak the soil, enabling easy uprooting of the plants. The roots of the uprooted plants were carefully and thoroughly washed to remove soil particles and debris, and then blotted dry with paper towels. The lengths of roots and shoots of each A. africana plant from the different treatments were measured using a meter ruler. The number of leaves and roots of each plant were counted. Fresh weights of the A. africana plants from the different treatments were obtained. Thereafter, the plants were oven dried at 60 °C for 48 h and their dry weights were recorded.

Effect of priming duration and priming treatments on in vitro germination of A. africana seeds.
The FGP values of all primed seeds were higher than those of non-primed seeds (control) ( Table 1).
Among the priming treatments and across all priming durations, the highest FGPs were recorded for seeds primed with 1.44 × 10 −3 M GA 3 , followed by A. africana seeds primed with 0.1 M KNO 3, and the lowest FGPs were recorded for hydro-primed seeds (  (Table 1). The highest MGR was recorded in seeds primed with 1.44 × 10 −3 M GA 3 for 24 h ( Table 1). The T 50 values for all concentrations of GA 3 -primed seeds decreased with an increase in priming duration (Table 1). There were no significant differences in T 50 values across all GA 3 priming concentrations and durations and across hydro-priming at all dura-  Effect of different priming treatments on ex vitro seed emergence. FGPs of GA3 -primed seeds were generally higher than those of halo-and hydro-primed seeds, with the highest overall FGP (80.0 ± 5.77%) recorded for 1.44 × 10 −3 M GA 3 primed seeds (Fig. 3a). Non-primed seeds had the lowest FGP (20.0% ± 2.89%), while the lowest FGP among primed seeds (45.0 ± 5.00%) was for the 1.0 M KNO 3 treatment (Fig. 3a). There were no significant differences in FGP among all concentrations of GA 3 and H 2 O and 0.1 M KNO 3 , but the FGPs of these treatments were significantly higher (p < 0.05) than FGPs for 0.5 M and 1.0 M KNO 3 primed and non-primed seeds (Fig. 3a). GI in hormo-primed seeds was significantly higher (p < 0.05) than that in all other treatments, except for hydro-primed seeds, with the highest GI at 3.76 ± 0.434 in the 1.44 × 10 −3 M GA 3 primed seeds (Fig. 3b). GI values decreased with an increase in KNO 3 concentration (Fig. 3b). The lowest GI value was recorded in the non-primed A. africana seeds (Fig. 3b). Fastest MGRs were attained in hormo-primed seeds, but did not significantly differ from MGRs of hydro-primed seeds. However, they did differ from MGRs of haloprimed and non-primed seeds (Fig. 3c). The lowest T 50 values were recorded in GA 3 -primed seeds, followed by hydro-primed seeds (Fig. 3d). In contrast, the longest germination periods with the highest T 50 values were for the halo-primed seeds (Fig. 3d). There were no significant differences in the T 50 values among all hormo-primed and hydro-primed seeds (Fig. 3d). T 50 for all hormo-and hydro-primed seeds was significantly shorter (p < 0.05) than that of all halo-primed and non-primed seeds (Fig. 3d).

Effect of different priming treatments on the early growth of A. africana. A. africana plant
growth was highest for seeds primed with GA 3 followed by KNO 3 and H 2 O, and lowest for non-primed seeds (Fig. 4). For all the growth parameters analyzed, plants from 2.89 × 10 −4 M GA 3 -primed seeds exhibited the best www.nature.com/scientificreports/ values, whereas plants from non-primed seeds registered the lowest values for all growth parameters ( Fig. 5a-f). All growth parameters of A. africana plants from halo-primed seeds decreased with increasing KNO 3 concentrations ( Fig. 5a-f). The highest average shoot length (333.3 ± 11.71 mm) of A. africana plants from 2.89 × 10 −4 M GA 3 -primed seeds did not vary significantly from the shoot lengths of plants from seeds primed with other concentrations of GA 3 and 0.1 M KNO 3 but significantly differed (p < 0.05) from the other treatments (Fig. 5a). The highest number of leaves (26.4 ± 1.15) in plants from 2.89 × 10 −4 M GA 3 -primed seeds was not significantly different from that of plants from other GA 3 -primed seeds, but was significantly higher (p < 0.05) than those from hydroand halo-primed seeds (Fig. 5b). Root lengths of plants from seeds primed with 2.89 × 10 −5 and 1.44 × 10 −3 M GA 3 , and 0.1 and 0.5 M KNO 3 did not differ significantly from the highest average root lengths (245.0 ± 15.82) of plants from 2.89 × 10 −4 M GA 3 -primed seeds, which was significantly higher (p < 0.05) than those from 1.0 M KNO 3 primed, hydro-primed, and non-primed seeds (Fig. 5c). The highest number of roots (24.8 ± 1.57) from 2.89 × 10 −4 M GA 3 -primed seeds did not vary significantly from those of other hormo-primed and all haloprimed seeds, but was significantly higher than those from hydro-primed and non-primed seeds (Fig. 5d). Fresh and dry weights of A. africana plants from all hormo-, halo-, and hydro-primed seeds were significantly higher than those from non-primed seeds (Fig. 5e, f). The fresh weights of plants from all primed seeds did not differ significantly (Fig. 5e), whereas the highest dry weight (1.98 ± 0.081 g) from 2.89 × 10 −4 M GA 3 primed seeds significantly differed from 0.5 M and 1.0 of KNO 3 and hydro-primed seeds (Fig. 5f).

Discussion
Temperature is a key factor that significantly affects germination 21,22 . Temperature directly influences imbibition and biochemical processes involved in germination that regulate metabolism, thus affecting germination rates and percentages 21 . Several studies have reported the effects of temperature on seed germination in different plants, including medicinal plants [23][24][25] . According to Baskin and Baskin 26 , the optimum temperature for many species is between 10 and 20 °C. In our study, low temperatures resulted in low FGPs and GIs for A. africana, and the values increased with temperature to optimal values of 65.0 ± 7.64% and 2.26 ± 0.223, respectively, at   www.nature.com/scientificreports/ 19.8 °C, and further decreased with increase in temperature. This trend has been observed in several other medicinal plant species showing low FPG at low and high temperatures, such as Nepeta binaludensis, Nepeta crassifolia, and Rubia tinctorum 23 . The percentage germination linearly increases with temperature until an optimum temperature is reached, and then sharply decreases 21 . Guo, et al. 21 further emphasizes that for most perennials, the favorable temperature for germination is 10-20 °C, and the optimum temperature for A. africana lies within this range. As observed, the lowest germination percentages occurred at the highest temperatures. High temperature inhibits germination of seeds in a number of species as it increases the endogenous levels of abscisic acid (ABA) by upregulating genes that biosynthesize ABA and downregulating genes associated with catabolism 27,28 . Furthermore, high temperatures decrease GA 3 content through repression of genes that biosynthesize GA 3 , thus inhibiting seed germination 27,28 . The thermoinhibitory effect of ABA has been demonstrated in a number of plant species, including Solanum lycopersicum 29 , and Pinus bungeana 21 . The MGR and T 50 values increased and decreased, respectively, with increasing temperature. This is presumably because the first phase of seed germination (imbibition) is greatly dependent on temperature and germination increases with increasing temperature 30 . Imbibition is a critical stage in seed germination, and the process is not only slowed down at low temperatures but also poses a great threat to cell membranes not adapted to low temperature 30 . Furthermore, the activities of some enzymes, such as dehydrogenases involved in the germination process, were found to increase with temperature 30 . The germination parameters for primed seeds for both in vitro and ex vitro experiments were better than those for non-primed seeds. Seed priming is a simple, safe and affordable technique for improving emergence, plant growth and yield [31][32][33] . Seed priming reduces the effect of abiotic stress during germination leading to higher emergence of seedling and vigorous establishment of seedlings [32][33][34] . In line with our observations, several studies previously confirmed that priming treatments greatly improved the germination parameters in a number of plants, such as Vicia faba L. 35 , and lentils 36 . Seed priming improves several physiological and metabolic processes, including activation of protective enzymes, such as catalase (CAT) and superoxide dismutase (SOD), and accumulation of osmoprotectants 37 . In a study by Armin, et al. 38 , KNO 3 treatment increased the FGP of sugar-beet seeds by up to 17.87% compared to the control. In another study, priming water melon seeds with KNO 3 and water increased FGP and GI 39 similar to the observations in our study. Improved germination parameters of seeds with KNO 3 priming were also observed for Glycine max 40 and Helianthus anuus 41 among others. In agreement with our findings for both in vitro and ex vitro investigations, GA 3 -priming of seeds from other plants, such as Medicago sativa 42 , and Hibiscus sabdariffa L 43 . is reported to greatly improve germination.
We observed that seed germination responses to priming were in the order GA 3 > KNO 3 > H 2 O. Similar to our observation, in a study on the medicinal plant Foeniculum vulgare, it was reported that GA 3 was also superior to other priming agents used, including KNO 3 44 . Tahaei, et al. 44 explained that GA 3 improves germination by upregulating α-amylase activity, eventually improving the metabolism of starch and sugar solubility. Furthermore, GA 3 activates embryo growth, reserve mobilization, and endosperm layer weakening, thus greatly improving germination 45,46 . Additionally, exogenous GA 3 was observed to greatly influence radicle protrusion in germinating Arabidopsis seeds 46 . In agreement with our results, Singh et al. 47 also observed that although both KNO 3 and H 2 O priming of seeds improved germination parameters, FGP for KNO 3 was better than that for H 2 O in cow pea. This could have been possible because KNO 3 supplied nitrate to the seeds and caused exosmosis that eliminated all germination inhibiting substances 47 . A similar finding was also reported for sorghum seeds primed with KNO 3 48 . Seed priming with KNO 3 is known to enhance germination, improve seedling growth, seedling vigor and drought tolerance through increased water imbibition, and activation of enzymes (amylases, xylanase, and dehydrogenases) and numerous ROS-scavenging antioxidants 32 . At the imbibition stage, seeds take up increased oxygen amount, resulting in accumulation of ROS shifting the redox state 49 . KNO 3 increases the activity of antioxidant enzymes such as SOD, CAT, ascorbate oxidase (AOX), and peroxidase (POX) in seedlings 49 .
Similar to our in vitro germination study, Damalas, et al. 35 reported that faba bean germination parameters were affected by priming duration. In their study, hydro-priming durations of 8 and 16 h had very high FGP and GI, which declined at longer priming durations of 24 and 48 h. Contrary to their findings, in our study, seeds hydro-primed for longer durations showed slightly improved germination, but for KNO 3 priming treatments, germination parameters declined at higher concentrations and longer treatment durations. The decline in germination in both our in vitro and ex vitro investigations with increasing concentrations of KNO 3 was possibly due to increasing external osmotic pressure, which affected imbibition by the seeds, leading to decreased FGP, decreased GI and MGR, and a longer T 50 duration. Oliveira, et al. 39 also reported decreased melon seed FGP and GI with increasing salt stress. Osmotic stress affects starch hydrolysis energy production, thus affecting germination 39,50 . Furthermore, in line with our observation, Ruttanaruangboworn, et al. 51 also reported a better germination response of Oryza sativa L. when primed with a lower concentration (1%) of KNO 3 than with KNO 3 at a higher concentration (2%). Generally, germination parameters improved with increasing GA 3 concentration, although there were no significant differences among the GA 3 -treated seeds for both in vitro and ex vitro investigations. Increasing the concentration of GA 3 improves the metabolic and physiological processes during germination. As in our study, priming of Capsicum annum L. seeds in 1.44 × 10 −3 M GA 3 resulted in the highest FGP of 85.98% 52 . Inconsistent with our findings, germination of Leymus chinensis seeds was best when primed with GA 3 at a concentration of 5.05 × 10 −5 M 53 . Such disparities could be attributed to differences in the species and seed conditions.
Comparing the in vitro and ex vitro germination parameters, the in vitro germination parameters were improved for both primed and non-primed A. africana seeds. Finch-Savage and Bassel 54 pointed out that soil is such an intricate environment that exerts considerable stress on germinating seeds and seedlings. Seeds and seedlings are therefore vulnerable to such complexity, including mechanical impedance 54 .
A. africana seed priming improved plant growth for all priming solutions, with all primed seeds recording increased plant growth compared to non-primed seeds. This observation is in agreement with findings from www.nature.com/scientificreports/ a number of previous studies 35,39,55,56 . In fact, Zhu, et al. 57 recorded increased root lengths, and fresh and dry stem weights of two Brassica napus L. varieties for all priming solutions when treated with five different priming agents that included GA 3 . Compared to non-primed seeds, priming causes increased cell division at the apical meristem of roots of seedlings, which eventually promotes growth and development 58 . Across all measured parameters, GA 3 -primed seeds produced plants with the highest growth compared to halo-and hydro-primed seeds. These observations were similar to those of previous research findings 39,56 . The superiority of GA 3 over halo-and hydro-priming could be because GA 3 breaks dormancy in seeds, promoting germination, increasing intermodal lengths and cell division in the cambial zone, and also causes an increase in leaf size 56,59 .
Similar to our findings, increased growth of plants from KNO 3 primed seeds has been previously reported 38,55,60,61 . Thejeshwini, et al. 56 pointed out that growth of plants from KNO 3 primed seeds was comparable to that of plants from GA 3 -primed seeds. Seed priming with KNO 3 greatly improved soybean plant height, dry weight, seedling shoot, and root lengths 62 . In another study, KNO 3 priming improved plant height, number of leaves, and leaf area among other growth parameters in rice 55 . Adnan, et al. 60 explained the increased growth observed in plants from KNO 3 primed seeds as a result of the nitrates that regulate growth and translocate photo-assimilates to specific plant parts, improving growth and yield. Hydro-priming improves the growth of a number of plants 35,60 . Hydro-priming increases shoot length, root length, and number of roots among other parameters in sorghum 60 . The shoots of hydro-primed seeds show higher amylase enzyme activity that enhances the hydrolysis of shoot transitory starch, providing more glucose and enabling more growth 58 .

Conclusion
In this study, in vitro germination level of non-primed A. africana seeds was low across all investigated temperatures. Hydro-, halo-, and hormonal priming greatly improved both in vitro germination and ex vitro emergence of A. africana seeds. For the in vitro setup, seeds primed with 1.44 × 10 −3 M GA 3 had the highest FGP and GI, and the shortest T 50 across all priming durations. Seeds primed in KNO 3 had better germination parameters at shorter priming durations compared to longer priming durations. Furthermore, the highest overall FGP was observed for seeds primed for 12 h in 1.44 × 10 −3 M GA 3 . Ex vitro seed emergence was significantly enhanced for seeds primed with GA 3 compared to non-primed seeds. In addition, the ex vitro A. africana seed emergence was significantly enhanced with a decrease in KNO 3 concentration. Priming A. africana seeds with H 2 O, KNO 3 , and GA 3 improved their growth parameters. After three months of treatment with 2.89 × 10 −4 M GA 3 , A. africana seeds produced plants with the longest shoot and root lengths, highest number of leaves and roots, and highest fresh and dry weights. In our study, we did not determine the base and ceiling temperatures for seed germination of A. africana, and we recommend further study in this regard. Seed priming of A. africana is a feasible approach to greatly improve germination. This is the first study investigating the effects of temperature and priming treatments on the germination and emergence of A. africana seeds.